U.S. patent application number 14/432793 was filed with the patent office on 2015-12-03 for nod2-dependent pathway of cytoprotection of stem cells.
This patent application is currently assigned to INSTITUT PASTEUR. The applicant listed for this patent is INSTITUT PASTEUR. Invention is credited to Giulia Nigro, Philippe Sansonetti.
Application Number | 20150343018 14/432793 |
Document ID | / |
Family ID | 47018933 |
Filed Date | 2015-12-03 |
United States Patent
Application |
20150343018 |
Kind Code |
A1 |
Sansonetti; Philippe ; et
al. |
December 3, 2015 |
NOD2-DEPENDENT PATHWAY OF CYTOPROTECTION OF STEM CELLS
Abstract
The present invention is directed to an agonist of NOD2 for use
in therapy for increasing the autonomous capacity of survival of
vertebrate adult stem cells, without loss of their capacity to
multiply and differentiate, and preferably the capacity of survival
of intestinal stem cells, especially in response to a stress. The
invention also concerns the use of an agonist of Nod2 for
increasing in vitro or ex vivo the autonomous capacity of survival,
without loss of multiplication and differentiation capacity of
mammalian adult stem cell. The invention also discloses different
media and support for mammalian adult stem cells. The invention
also concerns an in vitro screening process for identifying
molecules capable increasing, in response to a stress, the
autonomous capacity of survival, without loss of multiplication and
differentiation capacity of mammalian adult stem cells.
Inventors: |
Sansonetti; Philippe;
(Paris, FR) ; Nigro; Giulia; (Paris, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSTITUT PASTEUR |
Paris Cedex |
|
FR |
|
|
Assignee: |
INSTITUT PASTEUR
Paris
FR
|
Family ID: |
47018933 |
Appl. No.: |
14/432793 |
Filed: |
October 3, 2013 |
PCT Filed: |
October 3, 2013 |
PCT NO: |
PCT/EP2013/070620 |
371 Date: |
April 1, 2015 |
Current U.S.
Class: |
536/53 ; 435/26;
435/29 |
Current CPC
Class: |
C12N 2501/054 20130101;
G01N 33/502 20130101; A61K 38/14 20130101; C12N 5/068 20130101;
G01N 2333/904 20130101; A61K 38/05 20130101; G01N 33/5076 20130101;
G01N 33/5073 20130101 |
International
Class: |
A61K 38/14 20060101
A61K038/14; G01N 33/50 20060101 G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 3, 2012 |
EP |
12306208.5 |
Claims
1. Agonist of NOD2 for use in therapy for increasing the autonomous
capacity of survival of mammalian adult stem cells, without loss of
their capacity to multiply and differentiate.
2. The agonist of NOD2 for use according to claim 1, wherein said
adult stem cells are intestinal stem cells, mesenchymal stem cells,
hematopoietic stem cells or skin stem cells.
3. The agonist of NOD2 for use according to claim 1, wherein the
increase of the autonomous capacity of survival, without loss of
the capacity to multiply and differentiate, is in response to a
stress applied to the stem cells.
4. The agonist of NOD2 for use according to claim 3, wherein said
stress is a chemical stress, an oxidative stress, irradiation, a
pharmacological stress, a physical or physiopathological stress or
an infectious stress.
5. The agonist of NOD2 for use according to claim 3, wherein said
stress is induced by chemotherapy, by radiotherapy, by infection or
by tissue injury or dissection.
6. The agonist of NOD2 for use according to claim 3, wherein said
stress is induced by manipulation of said adult stem cells, during
harvest or implantation of these cells.
7. The agonist of NOD2 for use according to claim 6 during
transplantation of intestinal stem cells for treating intestinal
atrophy, crypt dysfunction or inflammatory bowel diseases, or
during transplantation of skin stem cell for accelerating repair of
skin and other epithelial wounds, or during bone marrow
transplantation.
8. Agonist of NOD2 for use in transplantation of mammalian adult
stem cells, for improving engraftment of said cells and tissue
regeneration.
9. The agonist of NOD2 for use according to claim 1, wherein said
agonist is Muramyl dipeptide
(N-acetylmuramyl-L-Alanyl-D-Isoglutamine) or a chemical derivative
thereof, including N-Glycolyl-Muramyl dipeptide, a 6-O-acyl
derivative of Muramyl dipeptide, muramyldipeptide with a C18 fatty
acid chain, MurNAc-Ala-D-isoGln-Lys,
MurNAc-Ala-D-isoGln-L-Lys(D-Asn) and murabutide
(N-Acetyl-muramyl-L-Alanyl-D-Glutamin-n-butyl-ester).
10-15. (canceled)
16. In vitro screening process for identifying molecules capable of
increasing, in response to a stress, the autonomous capacity of
survival of mammalian adult stem cells, without loss of their
capacity to multiply and differentiate, comprising: Providing
intestinal stem cells expressing NOD2 protein; Adding a test
molecule, and Detecting an increase in the number of organoids
formed in the presence of the test molecule, under appropriate
culture conditions.
Description
[0001] The present invention is concerned with mammalian stem
cells, and more specifically the cytoprotection of this type of
cells.
[0002] Stem cells are unspecialized cells that have two defining
properties, namely the ability to self-generate and the ability to
differentiate into multiple cell types. Whereas embryonic stem
cells can differentiate into all of the hundreds cell types of the
body (totipotency or pluripotency) and are apparently immortal in
culture, adult stem cells can differentiate into a limited range of
adult cells (multi-potency) and they are mortal, although they have
an extensive replication potential (Tower, 2012). Adult stem cells
are found in variety of tissues, including intestine, brain, bone
marrow, pancreas, liver, skin, skeletal muscle and kidney.
[0003] In different pathologies, some tissues or systems, like the
skin or the hematopoietic system, may be damaged, non-functional or
incapable of regeneration, as a result of dysfunctions at the stem
cell level. Likewise, stem cells may be damaged by therapy,
especially chemotherapy and radiotherapy applied for treating
cancers, adversely affecting their survival and regeneration
capacities and in fine jeopardizing the whole capacity of
self-renewal of the tissue.
[0004] In view of their multi-potency and their extensive and
sustained tissue renewal capacity, adult stem cells have been
widely studied for the last decades.
[0005] The use of adult stem cells in research and therapy is not
as the use of embryonic stem cells, because the production of adult
stem cells does not require the destruction of an embryo.
[0006] Their transplantation with a view to reconstituting a
damaged or absent tissue is practiced with promising results. As an
illustration, Stelzner and Chen have been able to make new
intestinal mucosa by transplanting mucosal stem cells from one rat
donor to another one (Stelzner and Chen, 2006). Transplantation of
hematopoietic stem cells to reconstitute the immune system,
especially in case of radiotherapy or myeloablation, is also
practiced in humans. Adult stem cell treatments have indeed been
successfully used for many years to treat leukemia and related
bone/blood cancers through bone marrow transplants. Adult stem
cells are also used in veterinary medicine to treat tendon and
ligament injuries in horses.
[0007] In order to obtain the best tissue or system repair results
after transplantation of stem cells, it is however imperative that
the stem cells be harvested and transplanted in favorable
conditions, ensuring the best survival rate, without loss of
self-renewal and differentiation potential, which is however a
challenging objective, in view of the stressful conditions imposed
by the cell manipulation consecutive to transplantation.
[0008] In addition to the above, stem cells are subjected to many
other stresses in vivo, that is extrinsic or intrinsic stress.
Stress reduction in stem cells is critical, due to their essential
role in tissue renewal and because of the risk of malignant
transformation.
[0009] Management of stress by adult stem cells is still poorly
understood for the time being and there is thus a need for means
for favoring stem cell survival when subjected to stress without
lost of their self renewal and differentiation capacities.
[0010] The present inventors have shown that adult stem cells
express the NOD2 protein, and that activation of this intracellular
receptor in adult stem cells ensures a cytoprotection of these
cells.
[0011] Nucleotide oligomerization domain (NOD) 2/Caspase activation
and recruitment domain (CARD) 15 belongs to the described family of
intracellular NOD-like receptor proteins (NLRs), which contain a
central nucleotide-binding site domain flanked on its C-terminal
side by a leucine-rich repeat domain and on its N-terminal side by
two CARD domains. The NOD2 protein is known to play an important
role in immune system function. The NOD2 protein is indeed
expressed and active in some types of immune system cells,
including monocytes and macrophages, and in dendritic cells. The
protein is also active in several types of epithelial cells,
including Paneth cells, which are found in the lining of the
intestine. These cells help defend the intestinal wall against
bacterial infection.
[0012] The NOD2 protein has several critical recognized functions
in immune defense against foreign invaders. The protein is a
cytosolic sensor for a conserved bacterially derived structure,
namely Muramyl di-peptide (N-acetylmuramyl-L-Alanyl-D-Isoglutamine
or MDP) and is capable of activating in response proinflammatory
signaling pathways, such as the NF-.kappa.B pathway. This protein
complex regulates also the activity of multiple other genes,
involved in control immune responses and inflammatory
reactions.
[0013] The critical importance of the NLR family of receptors in
inflammatory processes is further reinforced by the
characterization of Nod2 and Nalp3 as susceptibility genes for
different inflammatory disorders. The most prominent example is the
linkage between a frameshift mutation in the Nod2 gene and the
early onset of Crohn's disease.
[0014] Expression of Nod2 in mesenchymal stem cells of human
umbilical cord blood has been recently reported with an hypothetic
role in induction of some differentiation pathways (Kim et al,
2010).
[0015] Unexpectedly, the present inventors have shown that an
agonist of NOD2, namely MDP, is able to increase the number of
organoids formed from isolated intestinal stem cells, and that this
increase is NOD2-dependent, i.e. requires the presence of NOD2.
Moreover, they have shown that, at the level of the intestinal
crypts the nod2 gene is expressed not only by Paneth cells but also
by stem cells in a higher level than in Paneth cells. They finally
demonstrated that MDP has a cytoprotective effect on stem cells,
via the NOD2-pathway, especially in stress conditions, where MDP is
able to increase the survival capacity of stem cells and also to
increase the capacity of said cells to regenerate tissue.
[0016] The present invention is thus generally directed to
different uses, methods and compositions, based on the
cytoprotectant effect of NOD2-agonist on adult stem cells, and more
specifically vertebrate adult stem cells, preferably in mammalian
adult stem cells.
[0017] In a first aspect, the invention concerns an agonist of NOD2
for increasing the autonomous capacity of survival, multiplication
and differentiation of adult vertebrate stem cells, especially
mammalian adult stem cells, namely for use in therapy.
[0018] By NOD2 is meant the wild-type Nucleotide Oligomerization
Domain (NOD) 2 protein of the mammal in question, also called
Caspase Activation and Recruitment Domain (CARD) 15. This protein
is indeed highly conserved in different mammalian species. The
corresponding sequences can be found in the protein database for
different mammalian species or can be easily found by homology with
the already known sequences for the other species. The sequence of
human NOD2 protein is set forth in accession number AAG33677.1
(GI:11275614), and in SEQ ID No1. The murine NOD2 sequence is set
forth in SEQ ID No2, corresponding to Genbank accession number
AAN52478.1 (GI:30017199).
[0019] The increase in the capacity of survival, multiplication and
differentiation is particularly relevant in stressful conditions,
i.e. in conditions imposing a stress on the stem cells such that
their survival, or their capacity to multiply and differentiate, is
adversely affected. Indeed, when the stress imposed on the stem
cells is such that said cells die or become unable to multiply and
differentiate, their capacity of tissue regeneration is lost and
they thus become useless for the organism. The present invention
brings a protection to these stem cells, preventing at least in
part, these changes imposed by the stressful conditions, and thus
protecting the stem cells from dying or from becoming unable to
multiply and differentiate.
[0020] The agonist of NOD2 according to the present invention has
thus a cytoprotectant effect on the stem cells, preventing their
death and their loss of multiplication and differentiation
capacity.
[0021] According to the invention, by mammalian adult stem cells is
meant any mammalian cell fulfilling the functional definition: a
cell that has the potential to regenerate a plurality of tissues
over a lifetime, and which is not an embryonic stem cell, i.e.
which is not derived from the epiblast tissue of the inner cell
mass (ICM) of a blastocyst or earlier morula stage embryos. A stem
cell can also be defined as a multipotent cell with a high self
renewal capacity, with a telomerase activity and capable of
becoming quiescent; wherein multipotent or multipotential means
capable of differentiating into at least two cell types and
preferably more than two. Adult stem cells according to the
invention are thus to be distinguished from progenitor cells.
[0022] Preferably, a mammalian adult stem cell according to the
present invention is not a mesenchymal stem cell derived from Human
umbilical cord blood, which is an immature stem cell and not an
adult stem cell.
[0023] The present inventors have indeed observed that the novel
cytoprotection effect on adult stem cells, does not appear to be
obtained on embryonic stem cells.
[0024] In the context of the present invention, the term
"cytoprotectant effect" or "cytoprotection" of stem cells is thus
to be understood as an increase in survival rate or viability of
stem cells, accompanied by maintenance of their capacity of
multiplication and differentiation, which means the maintenance of
their potential of reconstituting a whole tissue. Stem cells
protected according to the invention thus remain multi-potent.
[0025] It is stressed that the cytoprotection provided by an
agonist of NOD2 according to the invention preferably applies at
the single cell level, i.e. adult stem cells have individually an
increased probability of remaining viable and being still capable
of multiplication and differentiation.
[0026] In this respect, it must be noted that the cytoprotection
provided by an agonist of NOD2 according to the invention is to be
distinguished from a direct induction of differentiation. Indeed,
the maintenance of the differentiation potential is different from
an induction of commitment in differentiation pathways with loss of
multipotency potential.
[0027] Cytoprotection, or increase in autonomous capacity of
survival, multiplication and differentiation, provided by
NOD2-agonist according to the invention, means that the treated
adult stem cells are maintained alive and that, concomitantly,
their capacity to self-renew and to replenish adult tissue is
preserved.
[0028] By self-renewal, it is to be understood the capacity for
division without altering the initial characteristics of the
cell.
[0029] Moreover, according to the invention, the cytoprotection
provided to stem cells by agonists of NOD2 is linked to an increase
of the stem cells' own capacity to overcome stressful conditions,
and not to the effect of further cells or factors to reduce
stress.
[0030] The mammalian stem cells according to the invention may be
any type of adult stem cells. Preferred types of adult stem cells
are intestinal stem cells, mesenchymal stem cells, hematopoietic
stem cells, skin stem cells and muscle stem cells. Other types of
stem cells also envisaged by the present invention are
adipose-derived stem cell, endothelial stem cell. Common myeloid
progenitors and common lymphoid progenitors, which are derived from
hematopoietic stem cells by differentiation, are thus not
hematopoietic stem cells.
[0031] Particularly preferred stem cells are NOD2 expressing stem
cells. According to a particularly preferred embodiment of the
invention, the adult stem cells are intestinal stem cells.
[0032] With regard to the stressful conditions imposed on the stem
cells according to the invention; they relate inter alia to any
conditions which do not correspond to the optimal conditions for
normal growth of the stem cells. Stress or stressful conditions
include conditions leading to loss of progenitor capacity and
conditions leading to cell death.
[0033] Stress can indeed take several forms, at the level of the
cell and at the level of the organism, it can be extrinsic or
intrinsic, or both, like aging. Extrinsic environmental stresses
include radiation (for example UV-radiation of X-ray radiation) and
xenobiotic toxins, like anticancer therapies. Biomechanical
stresses and physical stress are also encompassed by the present
invention. They comprise shear stress and hydrodynamic stress that
can damage the stem cells during manipulation.
[0034] According to preferred embodiments of the present invention,
the stress is a chemical stress, particularly an oxidative stress,
irradiation, or a pharmacological stress. A chemical stress can be
defined inter alia by imposing to the stem cells a particular
chemical environment that induces stress reactions and eventually
loss of progenitor capacity and/or cell death. An example of a
chemical stress is creation of conditions of oxidative stress in
the cellular milieu/environment. An oxidative stress can be defined
as exposure of cells to reactive oxygen species (ROS) produced by
dedicated enzymatic complexes by the cells of interest or
neighbouring cells under threat. Oxidative stress can be mimicked
by addition of molecules like H.sub.2O.sub.2. Irradiation is
preferably by UV or X-rays. A pharmacological stress can be defined
by the exposure to drugs like anti-cancer drugs, particularly
Doxorubicin that is particularly active on adult stem cells and is
able, as an intercalating agent to induce DNA breaks causing
induction of cell death pathways.
[0035] According to another embodiment of the present invention,
the stress applied to the stem cells is a physical or
physiopathological stress. By physical stress, it is understood
physical conditions leading to a stress, for example a shear stress
or hydrodynamic stress to the cell wall, likely to damage the cell.
Said physical stresses may especially be experienced during
manipulation of stem cells, either when harvested or extracted from
a donor or re-implanted or re-infused into a recipient, or during
any step of culture of said stem cells, including sampling,
sorting, seeding, dilution, change of medium, change of support,
etc. . . . .
[0036] A physiopathological stress can be defined as disturbances
of bodily function resulting from diseases. Examples of
physiopathological stresses likely to be applied to stem cells are
inter alia infections, particularly bacterial and viral infections,
or a stress generated by auto-immune response.
[0037] It is to be noted that both physical and pharmacological
conditions tested also cause the generation of ROS.
[0038] Another type of stress may be linked to deficiencies of
blood supply of the stem cells, linked to a defined pathology or to
a stroke.
[0039] According to preferred embodiments of the present invention,
the stress applied to the stem cells is induced by chemotherapy, by
radiotherapy, by infection or by tissue injury or dissection.
[0040] According to other preferred embodiments, the stress is
applied during any measures undertaken with a view to transplanting
said stem cells, especially any measures taken before or during
their harvest, extraction or collection, and any measures taken
before, during or after their re-implantation or re-infusion. The
transplantation may be autologous or non-autologous
transplantation. In case of non-autologous transplantation, the
stress applied to the stem cells may also be due to the immune
response of the organism in which they are implanted, or to the
drugs administered for diminishing this immune response.
[0041] A particularly preferred embodiment of the present invention
lies in the use of an agonist of NOD2 specifically during
transplantation of intestinal stem cells. Said transplantation of
intestinal stem cells may indeed be carried out for treating
intestinal atrophy or crypt dysfunction, or any other pathology
leading to a disruption of the intestinal homeostasis. The
intestinal atrophy may be linked to immunopathological diseases
such as celiac diseases; it may also be linked to pediatric
malnutrition in developing countries, in relation to epithelial
atrophy as part of the syndrome of environmental enteropathy. In
the frame of transplantation of organoids for treating Inflammatory
Bowel Diseases, comprising intestinal stem cells, an agonist of
NOD2 may also be used according to the invention. According to
another embodiment of the invention, the stem cells are skin stem
cells and the treatment with an agonist of NOD2 is during
transplantation of said stem cells. The transplantation of skin
stem cells may indeed be carried out for treating any skin or other
epithelial wounds, in order to accelerate skin or epithelial
regeneration.
[0042] According to a further embodiment of the invention, the stem
cells are hematopoiectic stem cells and the treatment with an
agonist of NOD2 is during bone marrow transplantation.
[0043] According to another embodiment of the present invention, an
agonist of NOD2 is for increasing in situ the autonomous capacity
of survival, multiplication and differentiation of mammalian
intestinal stem cells. This cytoprotection may be useful in
particular for intestinal epithelial repair in inflammatory bowel
diseases, in chemotherapies, in immunopathological diseases
inducing intestinal atrophy or in pediatric malnutrition in
relation to epithelial atrophy. In this cases indeed,
administration of an agonist of NOD2 is likely to preserve the
intestinal stem cells from the stress applied by the recited
pathologies or therapies, thus increasing their survival rate and
consequently their capacity to repair the intestine tissue or
repopulate the crypt with stem cells.
[0044] The present invention is also directed to an agonist of NOD2
for use in transplantation of mammalian adult stem cells, for
improving engraftment of said cells and tissue regeneration.
Indeed, as detailed above, transplantation corresponds to highly
stressful conditions for the stem cells, likely to induce their
death or incapacity to proliferate and differentiate.
[0045] For this embodiment also, the stem cells to be transplanted
are preferably NOD2-expressing stem cells. Particularly preferred
adult stem cells are inter alia adult intestinal, hematopoietic,
mesenchymal or skin cells, as mentioned above. The different
pathologies or diseases to be treated by transplantation of
mammalian adult stem cells have been detailed above.
[0046] For the different therapeutic applications described, the
agonist of NOD2 is preferably administered before or during the
application of the stressful conditions. It may advantageously be
applied (for example contacted with the stem cells) in the days
before initiation of the stress. For example, in case of harvest or
extraction of stem cells with a view to their transplantation, the
agonist of NOD2 is preferably applied beginning at least two days
before the day of harvest or extraction. In this case, it is
advantageously applied regularly, once or several times per day
until the intervention. The agonist may also be applied just before
the stress, or only concomitantly with the stressful
conditions.
[0047] According to the invention, it is also envisaged that the
agonist of NOD2 be first applied after the beginning of the
stressful conditions, and even after the return to optimal or
normal conditions. In this case, the purpose of the NOD2 agonist is
to increase the recovery rate or recovery capacity of the stem
cells.
[0048] With regard to the regimen and dosage, it will be defined by
the physician, taking account of the patient and the conditions
endangering the stem cells.
[0049] By agonist of NOD2 (or "NOD2 agonist"), it is to be
understood a molecule capable of interacting with the NOD2 receptor
intracellularly and capable of triggering a NOD2-dependant pathway.
A NOD2 dependent pathway may be the cytoprotectant effect according
to the invention, it may also be activation of the NF-.kappa.B
pathway or activation of the MAPK pathway. Preferably an agonist of
NOD2 according to the invention is a compound capable of
reproducing the cytoprotective effect on intestinal stem cells
reported in the experimental section below with
N-acetylmuramyl-L-Alanyl-D-Isoglutamine (Muramyl di-peptide). Such
a NOD2 agonist according to the invention is also referred to as a
cytoprotective NOD2 agonist, due to its cytoprotective action on
adult stem cells. A suitable system for testing whether a compound
is an agonist of NOD2 is for example the system described in
Girardin et al. (EMBO Repts, 2001), wherein activation of NOD2 is
followed by activation of the NF-.kappa.B pathway of signaling.
Another suitable system corresponds to the experimental work
detailed in the Example section below, using the number of
organoids formed from intestinal stem cells for determining and
assaying the cytoprotection provided by various compounds and thus
identifying NOD2 agonists according to the invention, namely
cytoprotective NOD2 agonists.
[0050] The well known natural agonist of NOD2 is MDP (Muramyl
dipeptide), more specifically
N-acetylmuramyl-L-Alanyl-D-Isoglutamine, having the formula (I)
below. MDP is the minimal bioactive peptidoglycan (PGN) motif
common to bacteria, either Gram+ or Gram- bacteria. The recognition
between MDP and NOD2 is highly stereospecific of the L-D isomer,
excluding any reaction to the D-D or L-L analogs. MDP can be
chemically synthesized or can be extracted from bacterial PGN.
[0051] Peptidoglycan itself is also to be considered as an agonist
of NOD2, through its capacity to release MDP. Any other compound
capable of releasing MDP in conditions suitable for stem cells
growth, is also to be considered as a NOD2 agonist. Prodrugs,
releasing MDP in vivo or in conditions close to in vivo conditions,
are encompassed in the definition of NOD2 agonist according to the
present invention.
[0052] The present invention also encompassed any derivative of
MDP, also capable of activating the NOD2 receptor. Such derivatives
of MDP can be inter alia constituent of peptidoglycan of certain
bacteria, differing slightly from the structure of MDP, however
maintaining the same or improved capacity to activate NOD2.
[0053] An example of derivative of MDP is represented by formula
(Ia) and any salts thereof, wherein:
[0054] R.sub.1 represents H, CH.sub.3 or an alkyl, aryl, arylalkyl,
heteroaryl, heteroarylalkyl, cycloalkyl, heterocycloalkyl, alkoxy,
heteroalkoxy, acyl or alkylamine moiety, having preferably between
1 to 24 carbon atoms, preferably between 1 to 12, preferably 1, 2,
3 or 4 carbon atoms. Preferably, R.sub.1 is H, CH.sub.3, OH,
CH.sub.2--OH, NH.sub.3 or phenyl.
[0055] R.sub.2 represents H, or an alkyl, aryl, arylalkyl, acyl,
heteroaryl, heteroarylalkyl, cycloalkyl, heterocycloalkyl, alkoxy,
heteroalkoxy, alkylamine or O-acyl moiety, having preferably
between 1 to 24 carbon atoms, preferably between 1 to 10 carbon
atoms. Preferably, R.sub.2 is H, COOH, CO--NH.sub.2 or CO--R, with
R being a C.sub.10-C.sub.20 alkyl, aryl, arylalkyl, heteroaryl or
heteroarylalkyl moiety.
[0056] Alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
cycloalkyl, heterocycloalkyl, alkoxy, heteroalkoxy, alkylamine and
O-acyl moieties have their usual definitions and possess preferably
less than 200 atoms or less than 100 atoms, even preferably less
than 60 atoms, or less than 40 atoms.
##STR00001##
[0057] A particularly preferred derivative is N-Glycolyl-Muramyl
dipeptide, formula (II), which can be found in mycobacteria and
differs from MDP only by the addition of N-Glycolyl moiety (R.sub.1
is CH.sub.2--OH).
##STR00002##
[0058] Example of another derivative is for example 6-O-acyl
derivatives of Muramyl dipeptide, especially
6-O-stearoyl-N-Acetyl-muramyl-L-Alanyl-D-Isoglutamine according to
formula (III) below.
[0059] Other derivatives are also encompassed by the present
invention, namely derivatives by addition of different moieties,
likely to improve the transport of the molecule intracellularly, or
to stabilize the molecule, or to limit any immune response.
##STR00003##
[0060] Other commercially available derivatives include the
Muramyldipeptide with a C18 fatty acid chain,
MurNAc-Ala-D-isoGln-Lys, naturally released by Lactobacillus
salivarus, MurNAc-Ala-D-isoGln-L-Lys(D-Asn), naturally released by
Lactobacillus acidophilus (Macho Fernandez et al., 2011), and
murabutide (N-Acetyl-muramyl-L-Alanyl-D-Glutamin-n-butyl-ester,
formula IV). Murabutide is a particularly preferred cytoprotective
NOD2 agonist, insofar as it is devoid of pyrogenic activity and
lacks somnogenic activity. Other derivatives can be envisaged by
the skilled person in the art without specific difficulties.
Potential derivatives of MDP are disclosed in Rubino et al,
2013.
[0061] Particularly preferred agonists of NOD2 are molecules
capable of passing through the cell membrane, either naturally or
through endogenous transporters, NOD2 being an intracellular
receptor.
[0062] The NOD2 agonist according to the invention may be
administered by any appropriate means, for example by injection or
by oral route, under any appropriate form. It can be administered
in association with one or several pharmacologically acceptable
carriers. It can also be associated with any other active
substances; it may also be administered separately, concomitantly
or sequentially with other active principles. It is also envisaged
according to the present invention to combine two different NOD2
agonists.
[0063] In a preferred embodiment of the present invention, the
mammalian stem cells to be treated by the agonist of NOD2 are human
stem cells. The therapeutic applications described above are indeed
well suited to treatment of human beings. Alternatively, the
mammalian stem cells may also be rat or murine stem cells, porcine
stem cells, simian stem cells, or any other type of stem cells
likely to represent a suitable model system for humans. According
to a further embodiment of the invention, the stem cells may be
from cat, dog, horse, pets or cattle.
[0064] It must be noted in this respect that, although the
sequences of NOD2 receptors from different mammals are slightly
different, it is expected that MDP is able to activate all these
different orthologs, insofar as MDP is a natural molecule and the
minimal bioactive peptidoglycan motif common to all bacteria and
thus naturally present in the gut of any mammals.
[0065] According to a second aspect, the present invention is more
specifically directed to different uses of agonists of NOD2,
especially in vitro or ex vivo uses of said agonists, during
culture or any type of manipulation of vertebrate adult stem cells,
preferably mammalian adult stem cells. Indeed, in view of the
ability of agonists of NOD2 as detailed with respect to the first
aspect of the invention, an agonist of NOD2, namely a
cytoprotective NOD2 agonist, can be used according to the invention
for increasing in vitro or ex vivo the autonomous capacity of
survival, multiplication and differentiation of mammalian adult
stem cells.
[0066] In case of in vitro or ex vivo culture of mammalian adult
stem cells, these stem cells can advantageously be stimulated by an
agonist of NOD2, in order to increase the viability of these cells
and to ensure that they do not lose their capacity to multiply and
differentiate during the culture.
[0067] This in vitro or ex vivo use according to the invention can
advantageously be carried out on an adult stem cell population,
preferably on an homogeneous population, i.e. containing only adult
stem cells, more particularly, on a clonal population; this use can
also be carried out on tissue explants of blood samples for
example, or on any tissue explants comprising adult stem cells.
[0068] The step of culture is a key step in all fundamental uses
and studies on stem cells, or example studies on their role and
function. It is indeed important that the stem cells not become
senescent (i.e not dying) during this culture step, especially
because stem cells are difficult to obtain, at least for primary
human stem cells, and because an important phenomenon studied on
these cells is aging, which necessitates very long culture periods
and thus requires that the stem cells remain viable and maintain
their self-renewal capacity. The step of in vitro culture is also
critical in the course of transplantation, after harvest or
extraction and before re-implantation or re-infusion.
[0069] Addition of agonist of NOD2 is particularly preferred when
the stem cells are experiencing a stress, when they are recovering
from a stress, or in preparation for application of a stress. The
present inventors have indeed shown that, by activation of the NOD2
pathways, through treatment with an agonist of NOD2, stem cells are
then protected against stressful conditions, especially their
mortality rate is less than the mortality rate of those stem cells
which are not treated with the agonist, and they keep their
capacity to multiply and differentiate at a better rate than
untreated cells. Activation of the NOD2 pathway can advantageously
be made in the hours preceding the stress, during the stress, or
even in the hours following the stress, in order to improve
recovery of the stem cells.
[0070] For this aspect of the invention, the type of adult stem
cells is the same as for the first aspect of the invention. The
adult stem cells are thus preferably adult intestinal stem cells,
mesenchymal stem cells, hematopoietic stem cells, muscle stem cells
or skin stem cells. Any other mammalian adult stem cells can also
be envisaged.
[0071] The stem cells likely to be treated by the agonist of NOD2
according to the invention are NOD2 expressing stem cells.
[0072] The populations of stem cells to be treated by an agonist of
NOD2 according to the invention are preferably entirely
homogeneous, i.e. they contain only stem cells. More particularly,
the populations may be clonal populations.
[0073] Particularly preferred stem cells are intestinal stem cells.
They have been shown recently capable of forming in vitro organoids
(Sato et al, 2009). Intestinal stem cells or organoids can be
transplanted into a recipient, in order to reconstitute a
functional mucosa. Transplantation of organoids can indeed be
carried out to treat different Inflammatory Bowel Diseases. It is
thus imperative that, during culture, the intestinal stem cells
remain viable and capable of differentiation, or that the number of
organoids formed from harvested stem cells be as high as
possible.
[0074] As detailed in the experimental section below, the present
inventors have shown that the formation of organoids is greatly
enhanced in presence of an agonist of NOD2. Indeed, the number of
organoids formed, when the intestinal stem cells are stimulated by
an agonist of NOD2, is greater than the number of organoids formed
in the absence of said agonist, and the number of dying cells is
less, when the stem cells are stimulated with an agonist of NOD2.
Moreover, after stimulation by NOD2, there are still stem cells
having the capacity to differentiate in all the lineages of the
tissue, i.e for intestinal stem cells, in all the four major
differentiated cell types: enterocytes, goblet cells,
enteroendocrine cells and Paneth cells.
[0075] The stress applied to these stem cells can be any type of
stress. Numerous potential stresses have been detailed with regard
to the first aspect of the invention and are also applicable to
this aspect of the invention, where the stem cells are cultured in
vitro or ex vivo. Namely, the stress applied to the stem cells can
be inter alia a chemical stress, an oxidative stress, a
pharmacological stress, a physical stress or an infectious
stress.
[0076] In vitro or ex vivo, the stress can be induced inter alia by
manipulation, culture, sampling or isolation of said adult stem
cells.
[0077] Examples of stresses more specific to the in vitro or ex
vivo culture are the stress applied to the culture during change of
media, the stress applied by sampling or sorting. Moreover,
specific manipulations can be carried out on the stem cells, likely
to induce an important stress. Namely, during in vitro or ex vivo
culture of stem cells, it is possible to transduce or transfect
these stem cells, e.g. for inserting a foreign resistance gene, or
to repair a defective gene. In case of gene therapy for example,
during the culture of the stem cells obtained from a patient, these
cells are transduced to correct the defective gene or to insert the
lacking gene, such that the "corrected" stem cells are then
re-implanted or re-infused into the patient.
[0078] The protection of adult stem cells provided by the agonist
of NOD2 according to the invention is recommended in any situation
requiring their purification and re-implantation, which are by
essence stressful conditions.
[0079] Agonists of NOD2 are particularly well adapted during these
manipulation steps, which are damaging the stem cells and generally
lead to the death of numerous stem cells.
[0080] Moreover, insofar as these stem cells are intended for
re-implantation or re-infusion, it is of utmost importance that the
cells not only survive, but also keep their potential to regenerate
the whole tissue, i.e. keep their capacity to multiply and
differentiate into a plurality of tissues. Indeed, stress or
stressful conditions include both conditions leading to loss of
progenitor capacity and conditions leading to cell death.
[0081] Potential agonists of NOD2 have been detailed with respect
to the first aspect of the invention. These molecules are also
suitable for this second aspect of the invention. Namely, a
particularly preferred agonist is the Muramyl-dipeptide
(N-acetylmuramyl-L-Alanyl-D-Isoglutamine), which is a constituent
of any bacteria.
[0082] Also envisaged according to the present invention are any
functional derivatives of MDP, especially derivatives by addition
of a functional moiety. This added moiety may increase the capacity
to get into the stem cells, to stabilize the compound, or for
example to render it compatible with any other components of the
cell culture medium.
[0083] Examples of potential derivatives are those detailed above,
especially compounds of formula (Ia), and preferably
N-Glycolyl-Muramyl dipeptide of formula (II) or a 6-O-acyl
derivative of Muramyl dipeptide, especially
6-O-stearoyl-N-Acetyl-muramyl-L-Alanyl-D-Isoglutamine. Commercial
available derivatives of MDP include Muramyldipeptide with a C18
fatty acid chain, MurNAc-Ala-D-isoGln-Lys, naturally released by
Lactobacillus salivarus, MurNAc-Ala-D-isoGln-L-Lys(D-Asn),
naturally released by Lactobacillus acidophilus, and
murabutide.
[0084] For the reasons already mentioned with respect to the first
aspect of the invention, the mammalian stem cells likely to be
treated by agonists of NOD2 are preferably human stem cells, either
with a view to further re-implanting or re-infusing them into human
beings, or with a view to undertaking studies regarding these
cells.
[0085] With a view to re-implanting or re-infusing stem cells,
which have been cultured in presence of an agonist of NOD2, the
safety of the agonist to be used should be verified. In this
respect, it must be noted that insofar as MDP is a natural
compound, present in the cell wall of bacteria, this compound is
particularly well suited, as this is a compound naturally present
in the mammalian body.
[0086] Other stem cells are murine stem cells or rat stem cells, as
these mammals can be easily used in studies. Other types of mammal
are also envisaged in the frame of the present invention. All these
preferred embodiments are detailed with respect to the different
other aspects of the invention.
[0087] All the preferred embodiments detailed in the section
dealing with the therapeutic applications of agonist of NOD2 in
cytoprotection of stem cells are also applicable in the context of
the different uses mentioned above.
[0088] According to a third aspect, the invention is also directed
to different products incorporating one or more agonists of NOD2,
suitable for the culture, manipulation, preservation or transport
of adult vertebrate stem cells, especially adult mammalian stem
cells. Such products are indeed particularly appropriate in order
to protect stem cells against mortality or loss of multiplication
and differentiation capacity, during any manipulation step.
[0089] The invention is especially directed to a cell culture
medium specifically dedicated to the culture of mammalian adult
stem cells, especially to intestinal stem cells, comprising an
agonist of NOD2 as cytoprotectant. It may also be a medium suitable
for freeze procedure, or for transport procedure, encapsulation,
transformation, transduction, long-term preservation, harvest,
re-implantation, re-infusion or for any other manipulation step of
these cells. Preferred mammalian adult stem cells according to this
aspect of the invention are the same as those recited for the other
aspect of the invention, i.e. any type of adult stem cells, inter
alia, intestinal stem cells, mesenchymal stem cells, hematopoietic
stem cells, skin stem cells and muscle stem cells. Other types of
stem cells also envisaged by the present invention are
adipose-derived stem cell, endothelial stem cell. The mammalian
adult stem cells are preferably human stem cells, or alternatively
rat or murine stem cells, porcine stem cells, simian stem cells, or
any other type of stem cells likely to represent a suitable model
system for humans.
[0090] The invention also concerns a medium specifically designed
for stem cells for retaining the undifferentiated state of these
cells, inter alia a medium devoid of differentiation factors.
Indeed, during transplantation procedure of stem cells, it is
generally imperative to retain the multipotency of the harvested
stem cells until reimplantation. In this case, an appropriate
medium or culture medium is for undifferentiated cells and does not
contain factor inducing differentiation of stem cells; such a
medium, devoid of factor inducing differentiation of stem cells;
advantageously comprises an agonist of NOD2 according to the
invention.
[0091] The medium can be liquid, semi-solid or solid. Nutrients and
other compounds may also be part of such a medium. Antibiotics can
also be added, at suitable concentrations.
[0092] The concentration of agonist of NOD2 in the medium,
especially in a culture medium, is preferably between 1 ng/ml and
100 .mu.g/ml, preferably between 10 ng/ml and 20 .mu.g/ml. Such a
medium or culture medium can be advantageously added to a
matrigel.
[0093] The present invention also concerns a cell support or a cell
matrix or matrigel, suitable for mammalian adult stem cells
culture, said support comprising an agonist of NOD2 as
cytoprotectant. According to another embodiment, this cell support
or a cell matrix or matrigel does not comprise factors inducing
differentiation of stem cells.
[0094] In the different embodiments described above regarding cell
culture medium, cell support, matrix or matrigel, the NOD2 agonist
is as described with respect to the other aspects of the invention.
NOD2 agonist is therefore preferably the muramyl-dipeptide
(N-acetylmuramyl-L-Alanyl-D-Isoglutamine), a derivative thereof,
especially derivatives of MDP of formula (Ia), inter alia
N-Glycolyl-Muramyl dipeptide. Example of such a derivative is for
example 6-O-acyl derivatives of Muramyl dipeptide, especially
6-O-stearoyl-N-Acetyl-muramyl-L-Alanyl-D-Isoglutamine according to
formula (III) above. Other commercially available derivatives
include, Muramyldipeptide with a C18 fatty acid chain,
MurNAc-Ala-D-isoGln-Lys (naturally released by Lactobacillus
salivarus), MurNAc-Ala-D-isoGln-L-Lys(D-Asn) (naturally released by
Lactobacillus acidophilus) and murabutide (formula IV). Other
derivatives can be envisaged by the skilled person in the art
without specific difficulties.
[0095] According to a fourth aspect, the present invention is
directed to different processes, taking advantage of the
cytoprotection provided to adult stem cells by agonists of
NOD2.
[0096] The present invention is inter alia directed to an in vitro
screening process for identifying molecules capable of increasing
the autonomous capacity of survival, multiplication and
differentiation of adult vertebrate stem cells, preferably adult
mammalian stem cells, especially in response to a stress. This
process preferably comprises a step of providing cells expressing
NOD2 protein, either naturally or after activation or transfection
of a transgene under an appropriate promoter. Preferably, the
expressing cells are from the same organism as the stem cells for
which a cytoprotectant is screened. The process comprises also a
step of adding a test molecule to said NOD2 expressing cell; this
addition step may be addition to the culture medium, or
introduction directly into the cells.
[0097] The process also comprises a step of detection of an
interaction between NOD2, expressed in the cells, and the test
molecule.
[0098] This interaction can be detected by any appropriate means
for measuring the activation of NOD2 in the cell. Depending on the
cell type, the activation of NOD2 can be detected for example by
IL-8 release detection, or by NF-.kappa.B pathway activation.
[0099] Any other pathways, known to be specific to NOD2 activation,
can be used for detecting the activation of NOD2 by a test
molecule. By this process, a test molecule can be identified, which
can then be used as cytoprotectant for stem cells.
[0100] The test molecule can be any kind of molecule, either
naturally occurring or chemically synthesized. Preferably, a test
molecule is a derivative of Muramyl-dipeptide
(N-acetylmuramyl-L-Alanyl-D-Isoglutamine), especially a molecule
according to formula (Ia), or a molecule capable of releasing MDP
in intracellular conditions, or capable of releasing a functional
equivalent of MDP.
[0101] A test molecule may also consist in the fusion of two
molecules, for example MDP with a further molecule. Said further
molecule may indeed either ease the transport inside stem cells, or
may induce transduction, or may help targeting specifically the
stem cells, or may add any function which could be beneficial.
[0102] In a further embodiment, the present invention is directed
to an in vitro screening method for identifying molecules capable
increasing the autonomous capacity of survival, without loss of
multiplication and differentiation capacities of mammalian adult
stem cells, especially in response to a stress.
[0103] According to this embodiment, a test molecule is added in
vitro to adult mammalian stem cells and the survival rate and
differentiation potential of the stem cells after addition of the
test molecule is monitored or assayed, in the following hours or
days. The survival rate and differentiation potential is
advantageously observed in condition allowing multiplication and
differentiation of the stem cells under optimal conditions.
Alternatively, the survival rate and maintaining of the
differentiation capacity are observed in response to a stress, for
example a chemical stress, an oxidative stress, a pharmacological
stress, a physical or physiopathological stress, irradiation,
infection, . . . .
[0104] According to a preferred embodiment of this screening
process, the adult stem cells are intestinal stem cells; preferably
expressing NOD2 protein. For this cell type, the survival rate and
differentiation potential is preferably assayed by determining the
number of organoids formed in the following days, for example after
one, two, three, four, five days or more. Examples of
implementation of this process are detailed in the experimental
section and the specific components to be added to the culture
medium to favor organoids formation, are detailed accordingly. The
stem cells can advantageously be seeded with Paneth cells, in order
to increase organoids formation.
[0105] The molecules to be tested according to this in vitro
process are the same as those detailed with regard to the preceding
in vitro process.
[0106] These screening processes may advantageously be carried out
for identifying derivatives of MDP, for example derivatives
according to formula (Ia), with improved functionality, or fewer
side effects, or simpler or less expensive process of synthesis, or
any other improved property. Alternatively, these screening
processes may also be carried out in order to screen a small
molecule bank, with chemical structures totally unrelated to MDP,
in order to identify novel classes of cytoprotective NOD2
agonists.
[0107] If an increase in the survival rate and maintenance of the
differentiation potential are observed, for example when an
increase in the number of organoids formed in presence of the
tested molecule is observed, the process may advantageously
comprises a step of controlling that said increase is
NOD2-dependent.
LEGEND OF FIGURES
[0108] FIG. 1: Growth curve of organoids.
[0109] FIG. 1A: FIG. 1A is the picture of an organoid, grown from
intestinal crypt. This is a structure delineated by a definite
monolayer of cells.
[0110] FIG. 1B: FIG. 1B represents difference between a viable and
a dying organoid. On the left a living organoid is shown, i.e. a
structure with a monolayer of cells surrounding a lumen, with new
crypts (protusions). On the right a dead organoid is shown, that
appears as a clump of dead cells. Bar 100 .mu.m.
[0111] FIG. 1C: FIG. 1C represents a typical growth curve of the
number of organoids upon stimulation by MDP, different other MAMPs,
and without stimulation (control). The curves depicted are the
results from one experiment, but are representative of the typical
growth curve.
FIG. 2: Effects of MAMPs on organoids formation: MDP induced higher
yield of organoids through Nod2 receptor recognition.
[0112] FIG. 2 illustrates the fold change in number of organoids,
formed from extracted crypts, depending on the MAMPs applied for
stimulation. Crypts were stimulated with soluble sonicated
peptidoglycan (PGN), muramyl-dipeptide (MDP), muramyl-tetrapeptide
(Tetra-dap), Escherichia coli lipopolysaccharide (LPS) (each at 10
.mu.g/ml), 10 ng/ml flagellin (Fla), 500 ng/ml synthetic
lipoprotein (Pam3CSK), or 1 .mu.M unmethylated CpG dinucleotides
(CpG). The average number of non-stimulated organoids (control) is
around 50, and the fold change value of the control is fixed to 1.
The fold change upon the various MAMPs applied is thus by reference
to non-stimulated organoids (Ctrl).
[0113] The results presented in FIG. 2 are the average of 5
different independent experiments. The fold change is measured at
day 4.
[0114] *** P<0.001, **P<0.01 Mann-Whitney.
[0115] As can be seen from this figure, PGN (peptidoglycan) and MDP
induce the formation of higher amount of organoids.
FIG. 3: proliferation assays. Cell Proliferation was Analyzed by
Cytometry after 4 Days of Culture.
[0116] FIG. 3A illustrates the rate of Ki-67 expression upon
different stimulation conditions, Ki-67 being a marker for
proliferative cells.
[0117] FIG. 3B illustrates the rate of EdU incorporation. After 4
days of culture, 10 .mu.M EdU were added for 2 hours and then the
organoids were recovered to monitor the EdU incorporation.
Representation profiles of MDP-treated (black) and non-treated
organoids (grey) are shown.
[0118] No differences in the rate of proliferation between treated
and un-treated organoids can be seen.
FIG. 4: Organoids from Nod1 and Nod2 KO mice.
[0119] FIG. 4 illustrates the fold change in number of organoids,
formed from extracted crypts, from mice knocked out for Nod1 or
Nod2 genes, upon different MAMPs stimulation conditions. By
definition, the fold change of the untreated organoids is set to 1,
and the fold changes upon the various stimulation conditions
applied are by reference to the non-stimulated organoids.
FIG. 5: Nod2 gene expression
[0120] FIG. 5 illustrates the Nod2 gene expression in
Lgr5-expressing stem cells, in Paneth cells and in the whole
crypts. The level of expression of Nod2 gene in whole crypts is
arbitrarily fixed to "1".
[0121] As can be inferred from the results of FIG. 5, the level of
nod2 mRNA is 5 times more in stem cells than in Paneth cells.
FIG. 6: Markers of stem cells and of Paneth cells.
[0122] The relative level of expression of different markers is
illustrated in FIG. 6. As for FIG. 5, the level of expression for
the different markers is arbitrarily fixed to "1" at the level of
the whole crypts.
[0123] As can be seen from this figure, Igr5, ascl2 and olfm4 which
are specific markers of intestinal stem cells are preferentially
expressed in intestinal stem cells extracted from crypts, whereas
CD24, IyzP and Defcr-rs1 are mainly expressed in Paneth cells, thus
confirming the nature of these cells.
FIG. 7: Markers of stem cells and of Paneth cells in WT and Nod2 KO
cells.
[0124] FIG. 7 illustrates results similar to those presented in
FIG. 6, for crypts extracted from WT mice and from mice KO for Nod2
gene.
FIG. 8: Single cells stimulation.
[0125] FIG. 8 depicts the average number of organoids formed per
well, depending on the cell types seeded in the well, without
stimulation, or under stimulation with MDP. 500 cells were seeded
per well in a 96-wells-plate.
[0126] Stem cells are either wild type or KO for the nod2 gene;
they are seeded in all wells.
[0127] Paneth cells are also either wild type or KO for the nod2
gene; They are present in the 3.sup.rd to 6.sup.th series of
experiments and absent in the 1.sup.st 2.sup.nd and 7.sup.th series
of experiments.
[0128] In the last (7.sup.th) series of experiments, Wnt is
added.
FIG. 9: Inducing stress to stem cells.
[0129] FIG. 9 illustrates the fold change in number of organoids,
formed from extracted crypts, upon stimulation with MDP or
un-treated, with or without application of a specific stress,
either Doxorubicin or H.sub.2O.sub.2. By definition, the fold
change of the untreated organoids is set to 1 in the absence of any
additional stress, and the fold changes upon the various
stimulation or stress conditions applied are by reference to the
non-stimulated non-stressed organoids.
[0130] Doxorubicin (1 .mu.M) or H.sub.2O.sub.2 (200 .mu.M) are
added during the embedding of the crypts in the matrigel.
Doxorubicin (DOX) belongs to the anthracycline class of
chemotherapeutic agents and works by intercalating DNA. Doxorubicin
has been shown to preferentially attack the cells at the bottom of
the crypt.
[0131] As can be seen from FIG. 9, MDP is able to protect stem
cells.
FIG. 10: LDH release.
[0132] FIG. 10 illustrates the percentage of LDH release from
crypts treated by different MAMPs, by reference to untreated crypts
(corresponding to the control, value set to 100).
[0133] As can be deduced from the results of FIG. 10, the
stimulation with PGN or MDP results in a higher viability of the
crypts, by reference to untreated crypts.
FIG. 11: Caspase-3 activation.
[0134] FIG. 11 depicts the percentage of caspase-3 positive crypts
in different conditions. The results are obtained after 6
hours.
[0135] Line 1: WT, non-treated mice. Line 2: Nod2 KO mice,
non-treated. Line 3: WT mice, treated with doxorubicin during 6
hours. Line 4: Nod2 KO mice, treated with doxorubicin during 6
hours. It can be observed that the absence of Nod2 induced more
death in the crypts.
FIG. 12: Organoids from Doxorubicin-treated mice.
[0136] FIG. 12 illustrates the fold change in number of organoids,
formed from extracted crypts from either WT or Nod2 KO mice, 72
hours after treatment with doxorubin, upon stimulation with MDP or
un-treated.
FIG. 13: Proliferation index.
[0137] FIG. 13 illustrates the percentage of EdU-positive cells per
crypt, in wild-type or Nod2 KO mice, after 2 hours of EdU
(5-ethynyl-2-deoxyuridine) treatment.
[0138] In WT mice, the crypts are prone to regenerate.
[0139] By definition, the fold change of the untreated organoids,
extracted from WT mice, is set to 1, and the fold changes upon the
various stimulation conditions are by reference to the
non-stimulated WT organoids.
[0140] As can be seen from this figure, upon stress (namely
doxorubicin), the stem cells are actively reactivated by the
MDP.
FIG. 14: Stimulated and non-stimulated organoids present the same
maximal size.
[0141] On the fourth day of culture, MDP-treated or control
organoids, were imaged. The area of the organoids was measured
using Axiovision software, and the values were plotted. In the FIG.
a box and whisker-plot shows the values obtained from one
representative experiment.
EXPERIMENTAL SECTION
Example 1
Material and Methods
Mice
[0142] B6.129P2-Lgr5.sup.tm1(cre/ESR1)Cle/J mice (Lgr5-EGFP)
(Barker et al., 2007) were purchased from The Jackson Laboratories.
Card4/Nod1 (Nod1 KO) mice were generated by Millenium
pharmaceuticals Boston. Card15/Nod2 deficient C57BL/6J (Nod2 KO)
(Barreau et al., 2007) were provided by J.-P. Hugot (Hopital Robert
Debre, Paris, France). All mice were kept in specific pathogen-free
conditions. When indicated the mice were injected intraperitoneally
with 200 .mu.g of EdU (Invitrogen) 2 hrs prior to sacrifice or with
10 .mu.g/kg doxorubicin hydrochloride (Sigma) and sacrificed at
indicated time points.
Crypts Isolation and Organoids Formation
[0143] Intestinal crypts were obtained following a protocol already
described (Sato et al., 2009). After isolation small intestines
were isolated, flushed with cold PBS followed by 0.3% bleach and
again PBS, opened longitudinally and the villi were removed. The
tissues were cutted in small pieces, washed in 5 mM PBS/EDTA for 5
min, and subsequently incubated in fresh 5 mM PBS/EDTA for 30 min
on ice. After severals vigourous shakings the crypts, released in
the supernatant, were passed through a cell strainer 70 .mu.m (BD
Falcon), spun down at 800 rpm, resuspended in DMEM and counted. The
volume corresponding to 500 crypts was distributed in two tubes per
condition, and spun down again. The pellet of crypts was or not
incubated with the following microbe-associated molecular patterns
(MAMPs) for 10 min at room temperature: 10 .mu.g/ml Soluble
sonicated peptidoglycan from E. coli K12 (PGN), 10 .mu.g/ml
Muramyl-dipeptide (MDP), 10 .mu.g/ml MDP rhodamine, 10 .mu.g/ml MDP
control (MDP-ctrl), 10 .mu.g/ml Lipopolysaccharide (LPS), 500 ng/ml
Lipoprotein (Pam3), 10 ng/ml Flagellin (Fla) (all purchased from
InvivoGen), 10 .mu.g/ml MurNAc-Tetra(DAP) (Tetra-dap) (kindly
provided by Dominique Mengin-Lecreulx). The crypts were embedded in
50 .mu.l of growth factor reduced matrigel (BD Biosciences) in a
24-well plates, incubate at 37.degree. C. 20 min and overload with
500 .mu.l of crypts medium (CM) as described previously (Sato et al
2009). Briefly, Advanced DMEM/F12 was supplemented with 100 U/ml
penicillin/streptomycin, 10 mM Hepes, 1.times.N2, 1.times.B27 (all
from Gibco), 50 ng/ml EGF (Peprotech), 100 ng/ml Noggin
(Peprotech), 500 ng/ml R-spondin1 (R&D). The medium was
exchanged every 4 days. To induce toxic stress to the organoids, 1
.mu.M Doxorubicin hydrochloride (from Sigma) or 200 .mu.M
H.sub.2O.sub.2 were added to the medium upon the embedding of the
crypts into the matrigel.
[0144] The number of organoids was evaluating with an inverted
microscope equipped with thermostatic chamber (temperature and
CO.sub.2).
Sorting and Culture of Single Cells
[0145] Isolated crypts from Lgr5-EGFP mice or Nod2KO mice were
incubated in HBSS w/o Ca.sup.+2 and Mg.sup.2+ supplemented with 0.3
U/ml Dispase (BD Biosciences), 0.8 U/ml DNase (Sigma) and 10 .mu.M
Y-27632 (Sigma) for 30 min at 37.degree. C.
[0146] Dissociated cells were washed with 1% PBS/BSA and stained
with CD24-APC antibody (clone M1/69 BioLegend) and EpCam Pe-Cy7
(clone G8.8 BioLegend) for 20 min at 4.degree. C., resuspended in
crypts medium supplemented with 1 .mu.M N-acetyl-L-cystein, 10
.mu.M Jag-1 (Anaspec) and 10 .mu.M Y-27632 (single cells
medium-SCM) and filtered with a 20 .mu.m mesh and analyzed with
MoFlo legacy (Beckman Coulter).
[0147] Singlets of viable epithelial cells were gated by negative
staining for propidium iodide and positive staining for EpCam. The
stem cells and Paneth cells were isolated respectively as
GFP.sup.hi+ population, and GFP.sup.-CD24.sup.hi+SSC.sup.hi
population.
[0148] Sorted cells were collected or in SCM for culturing
experiments or in RNAprotect Cell Reagent (Qiagen) for the RNA
extraction.
[0149] Stem cells and Paneth cells derived from Lgr5-EGFP or Nod2KO
mice were cultured alone or in different combinations. 500 cells
from each condition were re-suspended in 100 .mu.l of single cells
medium with or without MAMPs and, when indicated, with 100 ng/ml
wnt3 (R&D), seeded in 96-wells round-bottom plates and
incubated on ice for 15 min. The plate was spun at 300 g for 5 min
and 10 .mu.l of matrigel was added in each well. The number of
organoids was counting after 1 week.
Real time PCR
[0150] Total RNA was extracted with the RNeasy Micro Kit (Qiagen)
and the cDNA was made with SuperScript II Reverse Transcriptase
(Invitrogen) and oligo(dT)12-18 primer (Invitrogen) as recommended
by the suppliers. The following primers (purchased from Invitrogen)
were used:
TABLE-US-00001 Ascl2, (SEQ ID No3) 5'-AAGCACACCTTGACTGGTACG-3' and
(SEQ ID No4) 5'-AAGTGGACGTTTGCACCTTCA-3'; b2m, (SEQ ID No5)
5'-TCAGTCGTCAGCATGGCTCGC-3' and (SEQ ID No6)
5'-CTCCGGTGGGTGGCGTGAGTAT-3'; CD24, (SEQ ID No7)
5'-CGAGCTTAGCAGATCTCCACT-3' and (SEQ ID No8)
5'-GGATTTGGGGAAGCAGAAAT-3'; defcr-rs1, (SEQ ID No9)
5'-AAGAGACTAAAACTGAGGAGCAGC-3 and (SEQ ID No10)
5'-CGACAGCAGAGCGTGTA-3'; Lgr5, (SEQ ID No11)
5'-GACAATGCTCTCACAGAC-3' and (SEQ ID No12)
5'-GGAGTGGATTCTATTATTATGG-3'; LyzP, (SEQ ID No13)
5'-GAGACCGAAGCACCGACTATG-3' and (SEQ ID No14)
5'-CGGTTTTGACATTGTGTTCGC-3'; nod2, (SEQ ID No15)
5'-GGAGTGGAACAGCTGCGACCG-3' and (SEQ ID No16)
5'-GCACACTCAACCAGCGTGCG-3'; Olfm4, (SEQ ID No17)
5'-TGGGCAGAAGGTGGGACTGTGT-3' and (SEQ ID No18)
5'-CGGGAAAGGCGGTATCCGGC-3'.
[0151] Reactions were run on ABI 7900HT (Applied Biosystems) using
Power SYBR Green mix (Applied Biosystems) according with the
manufactures instructions. B2M RNA was used as an internal control,
and .DELTA..DELTA.CT (cycle threshold) values were calculated.
Immunohistochemistry
[0152] The tissue was processed as already described (Escobar et
al., 2011). The small intestines were flushed with PBS and everted
on 3-mm OD wooden skewers before fixation in 4% paraformaldehyde
(Electron Microscopy Sciences) for 2 h. Intestines were released
from the skewers by a longitudinal incision and rolled up using
wooden toothpicks, incubated for 2 h in 15% sucrose and overnight
at 4.degree. C. in 30% sucrose. Rolls were then embedded in OCT
compound (VWR) frozen in isopentane, cooled with dry ice, and
stored at -80.degree. C. Frozen blocks were cut with a thickness of
7 .mu.m using a CM 3050S cryostat (Leica), and sections were
collected on Superfrost plus slides (VWR) and stored at -80.degree.
C. Alternatively after the fixation the tissues were embedded in
paraffin using standard procedures.
[0153] Paneth cells were stained using a rabbit anti-Lysozyme
(1:500, Thermo) and Alexa fluor 568 as secondary antibody. DNA was
stained by DAPI (1 .mu.g/ml, Molecular Probes). The sections were
mounted with Prolong gold antifade (Invitrogen). Images were
acquired with confocal microscopy (Leica, SP5).
[0154] For the caspase-3 staining, tissue sections were
deparaffinized in xylene and then rehydrated in a series of
alcohols and PBS. Endogenous peroxidase activity was removed by
incubating the sections in 3% hydrogen peroxide for 10 min. Antigen
retrieval was done by boiling the sections in 10 mM sodium citrate
buffer for 20 min. Sections were permeabilized for 10 min with 0.5%
triton X-100, blocked with ultra V block (Labvision) 5 min and
incubated overnight with a rabbit cleaved Caspase-3 antibody (Cell
Signaling Technology, 1:200). Dako EnVision-system HRP anti-rabbit
(Dako) was then applied for 40 min and the reaction developed with
DAB (Dako). Slides were counterstained with Mayer hematoxylin and
mounted with Aquatex (Merck). The sections were imaging with Mirax
scanner (Zeiss).
Cytotoxicity Assay
[0155] Cultures of organoids were analyzed for the release of
lactate dehydrogenase (LDH) using Promega CytoTox 96 kit according
to provider's instructions. The absorbance was mesaured with the
Tecan Sunrise microplate reader and the ratio between the LDH
released into the supernatant and the total LDH in cell lysate was
measured.
Proliferation Assay
[0156] The supernatant was removed and the organoids were
dissociated using dispase (BD Bioscience). The cells were fixed
with 2% PFA, permeabilized with 0.1% triton X-100 and stained with
1:100 dilution of rabbit polyclonal anti Ki-67 antibody (Abcam) for
1 h. Alexa Fluor 647 was used as a secondary antibody. Data were
recorded using FACSCalibur flow cytometer (BD) and analyzed with
Flowjo software.
[0157] The proliferation of organoids was also evaluated by flow
cytometry using the Click-iT.RTM. EdU Alexa Fluor.RTM. 647 Flow
Cytometry Assay Kit (Invitrogen) according to provider's
instructions. Briefly, EdU (5-ethynyl-2-deoxyuridine) was added at
10 .mu.M for 2 h. Then the culture supernatant was removed, and the
organoids were dissociated by dispase (BD Bioscience). The cells
were fixed and permeabilized, and the click-iT reactions were
carried out. After the washes, the cells were analyzed with a
FACSCalibur flow cytometer (BD) and the data analyzed by Flowjo
software.
[0158] For the analysis of proliferation in the tissue, 10 mg/kg
EdU were injected IP and the animals were sacrificed after 2 h. The
Click-iT.RTM. EdU Alexa Fluor.RTM. 488 Imaging Kit was used on
paraffin slides that were processed according to the manufacturer's
instructions.
Scoring of Proliferative Index and Regenerative Crypts.
[0159] The proliferation index was calculated as a percentage of
EdU positive cells over the total number of cells in each crypt.
Data from five mice each group were obtained and at least 50 crypts
per section were examined for all histological parameters. A
regenerative crypt was defined as a crypt containing more than 10
EdU positive cells per crypt. Relative crypt size was determined as
crypt width x crypt height.
Statistics.
[0160] The descriptive statistical analysis was performed on
Graphpad Prism version 5 (Graphpad software Inc., San Diego,
Calif.). Results are expressed as mean.+-.SD. Statistical
comparisons were performed using the Mann-Whitney test U-test. A
P-value <0.05 was considered as significant.
Example 2
Results
Summary:
[0161] The present inventors have discovered a NOD2-dependant
pathway of cytoprotection of intestinal stem cells, in the presence
of muramyl-dipeptide, the best known NOD2-agonist. This is a
totally novel function of the NOD signaling pathway that
illuminates the pathogenesis of IBDs (inflammatory bowel diseases)
like Crohn disease, and offers a novel approach to maintain stem
cells alive in conditions of manipulation.
[0162] The intestinal mucosa surface is continuously exposed to a
complex and dynamic community of microorganisms, the microbiota.
These microbes establish symbiotic relationships with their hosts,
and this interaction plays a crucial role in the maintenance of
intestinal epithelial homeostasis. The bacteria present in the
lumen release microbial components, namely MAMPs for
microbe-associated molecular patterns, which access the intestinal
crypts, a sensitive region responsible for epithelial regeneration
where intestinal stem cells are located. Using a newly developed in
vitro method described by Clevers group (Sato et al., 2009), the
inventors cultured intestinal crypts organoids testing several
MAMPs. Starting from the first day of culture, they found a higher
number of organoids upon stimulation with bacterial peptidoglycan
(PGN), especially with MDP, a PGN subunit present in Gram positive
and Gram negative bacteria. On the contrary the inventors did not
observe any differences with other PGN motifs such as Tetra-dap.
This result is due to the fact that Tetradap and MDP are
respectively recognized by two different murine receptors, Nod1 and
Nod2 (Magalhaes et al., 2005; Girardin et al., 2003). Only Nod2
seems to be involved in the observed phenotype. Indeed comparing
the effect of PGN or MDP on crypts extracted from Nod1 or Nod2
deficient mice, the inventors observed the same effect seen in the
wt crypts in the Nod1 KO mice. As expected, organoids produced from
Nod2 KO mice were not affected by the presence of MDP.
[0163] Using Lgr5-EGFP-ires-creERT2 mice, the inventors were able
to sort intestinal stem cell (SC) and cultivate them to produce
organoids. When the Paneth cells were added to SC the rate of
organoids formation increased. Adding MDP to the medium of single
SC or into a co-culture of SC and Paneth cells the inventors always
observed an increase in numbers of organoids. It is known from the
literature that Nod2 is expressed at the bottom of the intestinal
crypts, at the level of Paneth cells. However, the higher survival
of organoids was not associated to the expression of Nod2 in Paneth
cells. In fact co-culturing Paneth cells isolated from Nod2 KO mice
with wt stem cells, the increase in the number of organoids due to
the presence of MDP was still present.
[0164] Analyzing the gene expression in those cells, the inventors
found that not only the Paneth cells express the nod2 gene but also
the Igr5 positive stem cells. Moreover nod2 gene is expressed 5
times more in SC compared to Paneth cells. This explains the direct
effect of MDP on stem cells.
[0165] Organoids produced upon stimulation with MDP had the same
rate of proliferation of the non-stimulated ones. Therefore MDP has
a cyto-protective effect on isolated intestinal crypts. In fact,
crypts stimulated with MDP release less LDH compared to untreated
organoids.
[0166] MDP shed by the luminal microbiota can thus interact with
the Nod2 receptor expressed by Paneth cell and stem cells. The
Paneth cells could be induced to release factors like EGF, TGF-a,
Wnt3 required to support the stem cells. On the other hand MDP
interacting directly with stem cells increases their survival.
Detailed Results:
[0167] Recently a culture system for intestinal crypts has been
described (Sato et al., 2009). Embedding isolated crypts in
matrigel and adding growth factors, leads to three-dimensional
structures, called organoids. Organoids recapitulate the
crypt-villus architecture, with an internal lumen, the different
epithelial lineages and stem cells located in surface protrusions,
corresponding to novel crypts.
Organoids Stimulation.
[0168] To evaluate the effect of bacterial products on intestinal
crypts the inventors grew organoids in the presence of different
purified or synthetic microbe-associated molecular patterns
(MAMPs). By definition, MAMPs are common to all the bacteria,
pathogenic and not pathogenic, and can be released from the
microbiota normally present in the gut (Garret et al., 2010). To
allow MAMPs to be entrapped in the lumen of organoids they added
them prior to embedding of the crypts into the matrigel. To check
that the MAMPs localized inside the organoid lumen, they used, for
instance, MDP-rhodamine in order to monitor the location of this
molecule upon stimulation. They observed that the red signal of the
MDP localized inside the organoid lumen.
[0169] To monitor if pattern recognition receptors (PRRs), upon
recognition of their ligands, affected the occurrence and
development of organoids, they assayed different MAMPs. Nod1 and
Nod2 receptors were stimulated using soluble sonicated
peptidoglycan (PGN); moreover they used Tetra-dap and MDP to
discriminate between the two Nod proteins, respectively Nod1 and
Nod2. Synthetic lipoprotein (Pam3), lipopolysaccharide (LPS), and
flagellin (Fla) and unmethylated CpG were also assayed to
respectively stimulate TLR2, TLR4, TLR5 or TLR9. Upon
MAMP-stimulation they monitored the number of formed organoids.
They considered as viable organoid a structure delineated by a
definite monolayer of cells (FIG. 1A). FIG. 1B are pictures showing
a viable organoid on the left and a dying organoid on the right.
Only the viable organoids are counted. A typical growth curve of
the number of viable organoids upon stimulation is shown in FIG.
1C. Starting from the first day they observed a higher number of
organoids in the conditions stimulated with PGN and with MDP,
compared to the others, and this difference was maintained, and
even increased over the time of the experiments.
[0170] They elected to monitor the number of organoids at day 4,
the time at which the medium was normally exchanged for long
culturing. Upon stimulation with PGN they observed around 5 times
more organoids compared with untreated cultures (FIG. 2). To
distinguish between the role of Nod1 and Nod2, they compared the
effect of their dedicated agonists and found the same effect seen
with PGN using MDP, the Nod2 agonist, but not with Tetra-dap, the
Nod1 agonist.
[0171] The average area of the MDP-treated organoids was smaller
than untreated organoids meaning that in presence of MDP more
crypts could survive and generate new organoids. Indeed, following
the crypt purification process, a very heterogeneous population of
explanted crypts with different degrees of viability was obtained,
thus introducing an asynchronous pattern of initiation of growth.
In presence of MDP more stem cells could survive giving new
organoids from these immature structures or also from isolated
single stem cells. Moreover, no difference was observed in the
maximum size of organoids compared to controls, indicating that
stimulation with MAMPs did not affect their growth rate. This point
is illustrated in FIG. 14. To confirm this observation, after 4
days of culture, treated or not treated organoids were tested for
the expression of Ki-67, a marker for proliferative cells and for
EdU (5-ethynyl-2'-deoxyuridine) incorporation. As shown in FIG. 3A
(Ki-67) and FIG. 3B (EdU) they did not observe any variation in the
rate of proliferation between organoids stimulated with PGN or MDP,
compared with untreated controls.
[0172] To further investigate the effect of PGN, they generated
organoids from Nod1 KO and Nod2 KO mice. After stimulation with PGN
and MDP, the number of organoids from Nod1 KO mice, was 3 to 5
times higher, compared with untreated ones (FIG. 4). To confirm
that the effect on the numbers of organoids was MDP-dependent, the
inactive form of MDP, the D-D isomer (MDP-ctrl) was tested. No
difference was observed with the control samples. They applied the
same stimulus to organoids from Nod2 KO mice and did not observe
any difference.
[0173] It has been shown that in the gut the Nod2 receptor is
present at the level of the crypts in mice (Kobayashi et al.,
2005). In human intestinal tissues it was demonstrated that Nod2 is
expressed in Paneth cells (Ogura et al., 2003; Lala et al., 2003).
Moreover an important role was shown for Paneth cells as main
constituent of the niche of Lgr5 stem cells in intestinal crypts
(Sato et al., 2010). To further investigate the expression of the
nod2 gene in intestinal crypts, stem cell and Paneth cells were
sorted, starting from Lgr5-EGFP knock-in mice. Epithelial cells
were selected using an anti-Epcam antibody. Stem cells were sorted
as GFP "highly positive", CD24 "medium" positive cells, while
Paneth cells were isolated as high-SSC and high-CD24
population.
Purity after Sorting.
[0174] To confirm the purity of the two cell populations, they
performed real time-PCR checking for expression of the respective
markers of stem cells and Paneth cells. B2M was used as an internal
control gene and .DELTA..DELTA.Ct values were calculated to obtain
relative expression compared to whole crypt expression. For the
stem cells they found high expression of Lgr5 (Barker et al.,
2007), Ascl2 (Van der Flier et al, 2009a) and Olfm4 (Van der Flier
et al, 2009b), and low expression of CD24. Paneth cells highly
expressed CD24 (Sato et al 2009), LyzP and defcr-rs1 (Garcia et
al., 2009) (FIG. 6). Then they analyzed stem cells and Paneth cells
for expression of the nod2 gene. Paneth cells expressed Nod2, as
previously reported, but in a lower level compared to stem cells,
that showed to have 5 times more Nod2 gene than Paneth cells (see
FIG. 5).
Sorting from Nod2 KO Mice.
[0175] Nod2 KO mice were generated by the disruption of the Nod2
gene with a EGFP cassette (Barreau et al., 2007). Cells that
normally express Nod2 thus became fluorescent. They decided to
evaluate the GFP signal at the level of intestinal crypts and
verified that the signal was generated by the stem cells and not by
the Paneth cells. They stained the Paneth cells with an
anti-lysozyme antibody, and observed that the green cells, were
interspersed between Paneth cells, exactly in the position of the
Lgr5 stem cells. Starting from this observation they decided to
extract crypts from Nod2 KO mice and to sort the GFP positive cells
to verify that those cells correspond to Lgr5-expressing stem
cells. Then they analyzed by RT-PCR the gene expression pattern of
the sorted cells, checking the respective markers of stem cells and
Paneth cells. They confirmed that GFP positive cells were
expressing the markers of stem cells at the level observed in
Lgr5-EGFP mice, and the same for the Paneth cells markers (FIG.
7).
Single Cells Stimulation.
[0176] Using the same sorting strategy used to isolate cells for
gene analysis, they recovered stem cells and Paneth cells from
Lgr5-EGFP and Nod2 KO mice. When GFP positive cells were sorted
from Nod2 KO mice and cultivated in appropriate medium, they
observed the formation of organoids, meaning that the cells had
properties of stem cells (FIG. 8). In this condition the average
number of organoids was about 1.5 fold lower compared with wt stem
cells. No differences were found when MDP was added to stem cells
originating from Nod2 KO mice. On the contrary, upon MDP
stimulation of wt stem cells, 2.5 more organoids were observed,
compared to the unstimulated condition (p<0.0001). Co-culturing
Paneth cells, derived from both wt and Nod2 KO mice, with wt stem
cells, or adding the wnt ligand to wt stem cells alone, the average
of organoids per well was around 3, a value similar to the one
obtained in MDP-treated wt stem cells. Adding MDP to a co-culture
associating wt stem cells and wt Paneth cells they observed again
an increase in the average of organoids, around 6 organoids per
well. The same result was obtained by co-culturing wt stem cells
and Nod2 KO Paneth cells, meaning that the increase in the number
of organoids upon stimulation was dependent on Nod2 expressed in
stem cells. Moreover upon MDP-stimulation, no differences were
observed in co-cultures associating Nod2 KO stem cells with wt or
Nod2 KO Paneth cells.
Inducing Stress.
[0177] To test if MDP had a protective effect on stem cells present
in the crypt, they decided to induce a stress targeted to
intestinal stem cells. As a drug they used 1 .mu.M doxorubicin
hydrochloride (dox), known to be toxic for the stem cell (Dekaney
et al., 2009), or 200 .mu.M H.sub.2O.sub.2 to induce an oxidative
stress.
[0178] After 4 days of treatment they counted 50% less organoids.
When the MDP was added to the crypts in presence of doxorubicin,
they were able to restore the normal growth of the organoids
maintaining the same ratio between stimulated and non-stimulated
organoids of 5 times. In H.sub.2O.sub.2 treated samples, the
increase in the number of organoids upon MDP-stimulation was still
present but in a lower ratio (FIG. 9).
Cytoprotection.
[0179] Finally to assess if the differences in the number of
organoids upon stimulation was dependent from a better survival of
the crypts in the presence of MDP, they tested organoid culture for
the release of lactate dehydrogenase (LDH). After 4 days of culture
in presence of PGN or MDP the organoids produced 50% less LDH to
non-stimulated crypts (FIG. 10).
Doxorubicin In Vivo.
[0180] To evaluate if the cytoprotective effect induced by MDP in a
Nod2-dependent pathway on stem cells, that they observed in vitro,
was also observed in vivo, they treated wt and Nod2 KO mice with
doxorubicin. Mice were injected intraperitoneally with 20 mg/kg
doxorubicin. Due to the fluorescent properties of doxorubicin, they
could observe in tissue sections that red signal of doxorubicin was
colocalizing with the GFP signal of the stem cells (in a Lgr5-EGFP
mouse). The percentage of crypts presenting at least one
caspase3-positive cell was counted. As shown in FIG. 11 upon
treatment with doxorubicin wt mice presented 30% of the crypts with
apoptotic cells, compare to 10% in the non-treated. In the Nod2 KO
mice the effect of doxorubicin caused an increase in the percentage
of apoptotic crypts, going from 14% in the non-treated to 54% in
the doxorubicin injected mice. To evaluate if upon doxorubicin
treatment the proliferation index was affected, the number of EdU
positive cells per crypts was counted and the percentage versus the
total number of cells in the crypts was calculated. Both wt and
Nod2 KO mice showed a decrease in the rate of proliferation
starting from 6 h upon the injection of doxorubicin until 24 h. The
Nod2 KO mice showed a low rate of proliferation in the crypts also
in the following days, instead at 48 h the percentage of
proliferative cells in the crypts of wt mice increased again until
72 h reaching almost the same rate observed in the non-treated mice
(FIG. 13).
[0181] In order to evaluate the capacity of stem cells to survive
and repair the damaged tissue upon doxorubicin-treatment, the
inventors used a modified clonogenic microcolony assay (Tustison et
al). At 72 h wt mice presented higher percentage of
regenerative/surviving crypts than in Nod2 KO mice and this was
associated with a bigger size of the crypts (Table 1).
[0182] After 72 h the intestinal crypts were extracted from the
doxorubicin treated mice, and tested for the capacity to form
organoids and to respond to MDP. As shown in FIG. 12 upon MDP
stimulation the number of organoids was increasing more than 10
times compare to non-stimulated samples.
TABLE-US-00002 TABLE 1 Comparison of intestinal regeneration in
wild-type and Nod2 KO mice after 72 hours upon doxorubicin
injection. wt 72 h dox Nod2 KO 72 h dox Percentage of regenerative
crypts 59.9 .+-. 6.4 33.9 .+-. 9.4** Relative size of crypts 3231
.+-. 105 2538 .+-. 180**
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Sequence CWU 1
1
1811040PRTHomo sapiens 1Met Gly Glu Glu Gly Gly Ser Ala Ser His Asp
Glu Glu Glu Arg Ala 1 5 10 15 Ser Val Leu Leu Gly His Ser Pro Gly
Cys Glu Met Cys Ser Gln Glu 20 25 30 Ala Phe Gln Ala Gln Arg Ser
Gln Leu Val Glu Leu Leu Val Ser Gly 35 40 45 Ser Leu Glu Gly Phe
Glu Ser Val Leu Asp Trp Leu Leu Ser Trp Glu 50 55 60 Val Leu Ser
Trp Glu Asp Tyr Glu Gly Phe His Leu Leu Gly Gln Pro 65 70 75 80 Leu
Ser His Leu Ala Arg Arg Leu Leu Asp Thr Val Trp Asn Lys Gly 85 90
95 Thr Trp Ala Cys Gln Lys Leu Ile Ala Ala Ala Gln Glu Ala Gln Ala
100 105 110 Asp Ser Gln Ser Pro Lys Leu His Gly Cys Trp Asp Pro His
Ser Leu 115 120 125 His Pro Ala Arg Asp Leu Gln Ser His Arg Pro Ala
Ile Val Arg Arg 130 135 140 Leu His Ser His Val Glu Asn Met Leu Asp
Leu Ala Trp Glu Arg Gly 145 150 155 160 Phe Val Ser Gln Tyr Glu Cys
Asp Glu Ile Arg Leu Pro Ile Phe Thr 165 170 175 Pro Ser Gln Arg Ala
Arg Arg Leu Leu Asp Leu Ala Thr Val Lys Ala 180 185 190 Asn Gly Leu
Ala Ala Phe Leu Leu Gln His Val Gln Glu Leu Pro Val 195 200 205 Pro
Leu Ala Leu Pro Leu Glu Ala Ala Thr Cys Lys Lys Tyr Met Ala 210 215
220 Lys Leu Arg Thr Thr Val Ser Ala Gln Ser Arg Phe Leu Ser Thr Tyr
225 230 235 240 Asp Gly Ala Glu Thr Leu Cys Leu Glu Asp Ile Tyr Thr
Glu Asn Val 245 250 255 Leu Glu Val Trp Ala Asp Val Gly Met Ala Gly
Pro Pro Gln Lys Ser 260 265 270 Pro Ala Thr Leu Gly Leu Glu Glu Leu
Phe Ser Thr Pro Gly His Leu 275 280 285 Asn Asp Asp Ala Asp Thr Val
Leu Val Val Gly Glu Ala Gly Ser Gly 290 295 300 Lys Ser Thr Leu Leu
Gln Arg Leu His Leu Leu Trp Ala Ala Gly Gln 305 310 315 320 Asp Phe
Gln Glu Phe Leu Phe Val Phe Pro Phe Ser Cys Arg Gln Leu 325 330 335
Gln Cys Met Ala Lys Pro Leu Ser Val Arg Thr Leu Leu Phe Glu His 340
345 350 Cys Cys Trp Pro Asp Val Gly Gln Glu Asp Ile Phe Gln Leu Leu
Leu 355 360 365 Asp His Pro Asp Arg Val Leu Leu Thr Phe Asp Gly Phe
Asp Glu Phe 370 375 380 Lys Phe Arg Phe Thr Asp Arg Glu Arg His Cys
Ser Pro Thr Asp Pro 385 390 395 400 Thr Ser Val Gln Thr Leu Leu Phe
Asn Leu Leu Gln Gly Asn Leu Leu 405 410 415 Lys Asn Ala Arg Lys Val
Val Thr Ser Arg Pro Ala Ala Val Ser Ala 420 425 430 Phe Leu Arg Lys
Tyr Ile Arg Thr Glu Phe Asn Leu Lys Gly Phe Ser 435 440 445 Glu Gln
Gly Ile Glu Leu Tyr Leu Arg Lys Arg His His Glu Pro Gly 450 455 460
Val Ala Asp Arg Leu Ile Arg Leu Leu Gln Glu Thr Ser Ala Leu His 465
470 475 480 Gly Leu Cys His Leu Pro Val Phe Ser Trp Met Val Ser Lys
Cys His 485 490 495 Gln Glu Leu Leu Leu Gln Glu Gly Gly Ser Pro Lys
Thr Thr Thr Asp 500 505 510 Met Tyr Leu Leu Ile Leu Gln His Phe Leu
Leu His Ala Thr Pro Pro 515 520 525 Asp Ser Ala Ser Gln Gly Leu Gly
Pro Ser Leu Leu Arg Gly Arg Leu 530 535 540 Pro Thr Leu Leu His Leu
Gly Arg Leu Ala Leu Trp Gly Leu Gly Met 545 550 555 560 Cys Cys Tyr
Val Phe Ser Ala Gln Gln Leu Gln Ala Ala Gln Val Ser 565 570 575 Pro
Asp Asp Ile Ser Leu Gly Phe Leu Val Arg Ala Lys Gly Val Val 580 585
590 Pro Gly Ser Thr Ala Pro Leu Glu Phe Leu His Ile Thr Phe Gln Cys
595 600 605 Phe Phe Ala Ala Phe Tyr Leu Ala Leu Ser Ala Asp Val Pro
Pro Ala 610 615 620 Leu Leu Arg His Leu Phe Asn Cys Gly Arg Pro Gly
Asn Ser Pro Met 625 630 635 640 Ala Arg Leu Leu Pro Thr Met Cys Ile
Gln Ala Ser Glu Gly Lys Asp 645 650 655 Ser Ser Val Ala Ala Leu Leu
Gln Lys Ala Glu Pro His Asn Leu Gln 660 665 670 Ile Thr Ala Ala Phe
Leu Ala Gly Leu Leu Ser Arg Glu His Trp Gly 675 680 685 Leu Leu Ala
Glu Cys Gln Thr Ser Glu Lys Ala Leu Leu Arg Arg Gln 690 695 700 Ala
Cys Ala Arg Trp Cys Leu Ala Arg Ser Leu Arg Lys His Phe His 705 710
715 720 Ser Ile Pro Pro Ala Ala Pro Gly Glu Ala Lys Ser Val His Ala
Met 725 730 735 Pro Gly Phe Ile Trp Leu Ile Arg Ser Leu Tyr Glu Met
Gln Glu Glu 740 745 750 Arg Leu Ala Arg Lys Ala Ala Arg Gly Leu Asn
Val Gly His Leu Lys 755 760 765 Leu Thr Phe Cys Ser Val Gly Pro Thr
Glu Cys Ala Ala Leu Ala Phe 770 775 780 Val Leu Gln His Leu Arg Arg
Pro Val Ala Leu Gln Leu Asp Tyr Asn 785 790 795 800 Ser Val Gly Asp
Ile Gly Val Glu Gln Leu Leu Pro Cys Leu Gly Val 805 810 815 Cys Lys
Ala Leu Tyr Leu Arg Asp Asn Asn Ile Ser Asp Arg Gly Ile 820 825 830
Cys Lys Leu Ile Glu Cys Ala Leu His Cys Glu Gln Leu Gln Lys Leu 835
840 845 Ala Leu Phe Asn Asn Lys Leu Thr Asp Gly Cys Ala His Ser Met
Ala 850 855 860 Lys Leu Leu Ala Cys Arg Gln Asn Phe Leu Ala Leu Arg
Leu Gly Asn 865 870 875 880 Asn Tyr Ile Thr Ala Ala Gly Ala Gln Val
Leu Ala Glu Gly Leu Arg 885 890 895 Gly Asn Thr Ser Leu Gln Phe Leu
Gly Phe Trp Gly Asn Arg Val Gly 900 905 910 Asp Glu Gly Ala Gln Ala
Leu Ala Glu Ala Leu Gly Asp His Gln Ser 915 920 925 Leu Arg Trp Leu
Ser Leu Val Gly Asn Asn Ile Gly Ser Val Gly Ala 930 935 940 Gln Ala
Leu Ala Leu Met Leu Ala Lys Asn Val Met Leu Glu Glu Leu 945 950 955
960 Cys Leu Glu Glu Asn His Leu Gln Asp Glu Gly Val Cys Ser Leu Ala
965 970 975 Glu Gly Leu Lys Lys Asn Ser Ser Leu Lys Ile Leu Lys Leu
Ser Asn 980 985 990 Asn Cys Ile Thr Tyr Leu Gly Ala Glu Ala Leu Leu
Gln Ala Leu Glu 995 1000 1005 Arg Asn Asp Thr Ile Leu Glu Val Trp
Leu Arg Gly Asn Thr Phe 1010 1015 1020 Ser Leu Glu Glu Val Asp Lys
Leu Gly Cys Arg Asp Thr Arg Leu 1025 1030 1035 Leu Leu 1040
21013PRTMus musculus 2Met Cys Ser Gln Glu Glu Phe Gln Ala Gln Arg
Ser Gln Leu Val Ala 1 5 10 15 Leu Leu Ile Ser Gly Ser Leu Glu Gly
Phe Glu Ser Ile Leu Asp Trp 20 25 30 Leu Leu Ser Trp Asp Val Leu
Ser Arg Glu Asp Tyr Glu Gly Leu Ser 35 40 45 Leu Pro Gly Gln Pro
Leu Ser His Ser Ala Arg Arg Leu Leu Asp Thr 50 55 60 Val Trp Asn
Lys Gly Val Trp Gly Cys Gln Lys Leu Leu Glu Ala Val 65 70 75 80 Gln
Glu Ala Gln Ala Asn Ser His Thr Phe Glu Leu Tyr Gly Ser Trp 85 90
95 Asp Thr His Ser Leu His Pro Thr Arg Asp Leu Gln Ser His Arg Pro
100 105 110 Ala Ile Val Arg Arg Leu Tyr Asn His Val Glu Ala Met Leu
Glu Leu 115 120 125 Ala Arg Glu Gly Gly Phe Leu Ser Gln Tyr Glu Cys
Glu Glu Ile Arg 130 135 140 Leu Pro Ile Phe Thr Ser Ser Gln Arg Ala
Arg Arg Leu Leu Asp Leu 145 150 155 160 Ala Ala Val Lys Ala Asn Gly
Leu Ala Ala Phe Leu Leu Gln His Val 165 170 175 Arg Glu Leu Pro Ala
Pro Leu Pro Leu Pro Tyr Glu Ala Ala Glu Cys 180 185 190 Gln Lys Phe
Ile Ser Lys Leu Arg Thr Met Val Leu Ala Gln Ser Arg 195 200 205 Phe
Leu Ser Thr Tyr Asp Gly Ser Glu Asn Leu Cys Leu Glu Asp Ile 210 215
220 Tyr Thr Glu Asn Ile Leu Glu Leu Arg Thr Glu Val Gly Thr Ala Gly
225 230 235 240 Ala Leu Gln Lys Ser Pro Ala Ile Leu Gly Leu Glu Asp
Leu Phe Asp 245 250 255 Thr His Gly His Leu Asn Arg Asp Ala Asp Thr
Ile Leu Val Val Gly 260 265 270 Glu Ala Gly Ser Gly Lys Ser Thr Leu
Leu Gln Arg Leu His Leu Leu 275 280 285 Trp Ala Thr Gly Arg Ser Phe
Gln Glu Phe Leu Phe Ile Phe Pro Phe 290 295 300 Ser Cys Arg Gln Leu
Gln Cys Val Ala Lys Pro Leu Ser Leu Arg Thr 305 310 315 320 Leu Leu
Phe Glu His Cys Cys Trp Pro Asp Val Ala Gln Asp Asp Val 325 330 335
Phe Gln Phe Leu Leu Asp His Pro Asp Arg Val Leu Leu Thr Phe Asp 340
345 350 Gly Leu Asp Glu Phe Lys Phe Arg Phe Thr Asp Arg Glu Arg His
Cys 355 360 365 Ser Pro Ile Asp Pro Thr Ser Val Gln Thr Leu Leu Phe
Asn Leu Leu 370 375 380 Gln Gly Asn Leu Leu Lys Asn Ala Cys Lys Val
Leu Thr Ser Arg Pro 385 390 395 400 Asp Ala Val Ser Ala Leu Leu Arg
Lys Phe Val Arg Thr Glu Cys Gln 405 410 415 Leu Lys Gly Phe Ser Glu
Glu Gly Ile Gln Leu Tyr Leu Arg Lys His 420 425 430 His Arg Glu Pro
Gly Val Ala Asp Arg Leu Ile Gln Leu Ile Gln Ala 435 440 445 Thr Ser
Ala Leu His Gly Leu Cys His Leu Pro Val Phe Ser Trp Met 450 455 460
Val Ser Arg Cys His Arg Glu Leu Leu Leu Gln Asn Arg Gly Phe Pro 465
470 475 480 Thr Thr Ser Thr Asp Met Tyr Leu Leu Ile Leu Gln His Phe
Leu Leu 485 490 495 His Ala Ser Pro Pro Asp Ser Ser Pro Leu Gly Leu
Gly Pro Gly Leu 500 505 510 Leu Gln Ser Arg Leu Ser Thr Leu Leu His
Leu Gly His Leu Ala Leu 515 520 525 Arg Gly Leu Ala Met Ser Cys Tyr
Val Phe Ser Ala Gln Gln Leu Gln 530 535 540 Ala Ala Gln Val Asp Ser
Asp Asp Ile Ser Leu Gly Phe Leu Val Arg 545 550 555 560 Ala Gln Ser
Ser Val Pro Gly Ser Lys Ala Pro Leu Glu Phe Leu His 565 570 575 Ile
Thr Phe Gln Cys Phe Phe Ala Ala Phe Tyr Leu Ala Val Ser Ala 580 585
590 Asp Thr Ser Val Ala Ser Leu Lys His Leu Phe Ser Cys Gly Arg Leu
595 600 605 Gly Ser Ser Leu Leu Gly Arg Leu Leu Pro Asn Leu Cys Ile
Gln Gly 610 615 620 Ser Arg Val Lys Lys Gly Ser Glu Ala Ala Leu Leu
Gln Lys Ala Glu 625 630 635 640 Pro His Asn Leu Gln Ile Thr Ala Ala
Phe Leu Ala Gly Leu Leu Ser 645 650 655 Gln Gln His Arg Asp Leu Leu
Ala Ala Cys Gln Ile Ser Glu Arg Val 660 665 670 Leu Leu Gln Arg Gln
Ala Arg Ala Arg Ser Cys Leu Ala His Ser Leu 675 680 685 Arg Glu His
Phe His Ser Ile Pro Pro Ala Val Pro Gly Glu Thr Lys 690 695 700 Ser
Met His Ala Met Pro Gly Phe Ile Trp Leu Ile Arg Ser Leu Tyr 705 710
715 720 Glu Met Gln Glu Glu Gln Leu Ala Gln Glu Ala Val Arg Arg Leu
Asp 725 730 735 Ile Gly His Leu Lys Leu Thr Phe Cys Arg Val Gly Pro
Ala Glu Cys 740 745 750 Ala Ala Leu Ala Phe Val Leu Gln His Leu Gln
Arg Pro Val Ala Leu 755 760 765 Gln Leu Asp Tyr Asn Ser Val Gly Asp
Val Gly Val Glu Gln Leu Arg 770 775 780 Pro Cys Leu Gly Val Cys Thr
Ala Leu Tyr Leu Arg Asp Asn Asn Ile 785 790 795 800 Ser Asp Arg Gly
Ala Arg Thr Leu Val Glu Cys Ala Leu Arg Cys Glu 805 810 815 Gln Leu
Gln Lys Leu Ala Leu Phe Asn Asn Lys Leu Thr Asp Ala Cys 820 825 830
Ala Cys Ser Met Ala Lys Leu Leu Ala His Lys Gln Asn Phe Leu Ser 835
840 845 Leu Arg Val Gly Asn Asn His Ile Thr Ala Ala Gly Ala Glu Val
Leu 850 855 860 Ala Gln Gly Leu Lys Ser Asn Thr Ser Leu Lys Phe Leu
Gly Phe Trp 865 870 875 880 Gly Asn Ser Val Gly Asp Lys Gly Thr Gln
Ala Leu Ala Glu Val Val 885 890 895 Ala Asp His Gln Asn Leu Lys Trp
Leu Ser Leu Val Gly Asn Asn Ile 900 905 910 Gly Ser Met Gly Ala Gln
Ala Leu Ala Leu Met Leu Glu Lys Asn Lys 915 920 925 Ser Leu Glu Glu
Leu Cys Leu Glu Glu Asn His Ile Cys Asp Glu Gly 930 935 940 Val Tyr
Ser Leu Ala Glu Gly Leu Lys Arg Asn Ser Thr Leu Lys Phe 945 950 955
960 Leu Lys Leu Ser Asn Asn Gly Ile Thr Tyr Arg Gly Ala Glu Ala Leu
965 970 975 Leu Gln Ala Leu Ser Arg Asn Ser Ala Ile Leu Glu Val Trp
Leu Arg 980 985 990 Gly Asn Thr Phe Ser Leu Glu Glu Ile Gln Thr Leu
Ser Ser Arg Asp 995 1000 1005 Ala Arg Leu Leu Leu 1010 321DNAMus
musculus 3aagcacacct tgactggtac g 21421DNAMus musculus 4aagtggacgt
ttgcaccttc a 21521DNAMus musculus 5tcagtcgtca gcatggctcg c
21622DNAMus musculus 6ctccggtggg tggcgtgagt at 22721DNAMus musculus
7cgagcttagc agatctccac t 21820DNAMus musculus 8ggatttgggg
aagcagaaat 20924DNAMus musculus 9aagagactaa aactgaggag cagc
241017DNAMus musculus 10cgacagcaga gcgtgta 171118DNAMus musculus
11gacaatgctc tcacagac 181222DNAMus musculus 12ggagtggatt ctattattat
gg 221321DNAMus musculus 13gagaccgaag caccgactat g 211421DNAMus
musculus 14cggttttgac attgtgttcg c 211521DNAMus musculus
15ggagtggaac agctgcgacc g 211620DNAMus musculus 16gcacactcaa
ccagcgtgcg 201722DNAMus musculus 17tgggcagaag gtgggactgt gt
221820DNAMus musculus 18cgggaaaggc ggtatccggc 20
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